The Polymerase Chain Reaction (PCR) is a process to amplify DNA by replicating a certain part of DNA repeatedly, which produces identical DNA in large amount from an infinitesimal amount of DNA.
Through the Polymerase Chain Reaction, it is possible to selectively amplify a desired part of DNA collected from huge DNA like genome DNA. The Polymerase Chain Reaction comprises 3 steps as follows.
1) DNA Denaturation
Double-stranded DNA (dsDNA) is separated into single-stranded DNA (ssDNA) by heating DNA at between 90° C. to 96° C. Though the double-stranded DNA (dsDNA) can be denatured to single-stranded (ssDNA) more easily as they are heated at more high temperature, it is appropriate to be denatured at 94° C. in general because the activity of Taq. DNA Polymerase may be decreased at high temperature.
2) Primer Binding (Annealing)
The annealing process which binds the single stranded DNA with a primer, is, in general, proceeded 50° C. to 65° C. It can be possible to reduce the generation of non-specific product by means of raising the annealing temperature, particularly in case that non-specific product is serious.
3) DNA Replication (Polymerizaion, Extension)
The DNA replication is proceeded at 70° C. to 74° C. by the operation of Taq polymerase. In the case that the size of DNA to be amplified is large or the density of the cDNA is low, it is desirable to extend the replication time. The replication can be proceeded sufficiently by allowing a time of almost one (1) minute per 1 KB since Taq. Polymerase can replicate DNA of the length of 2,000 to 4,000 base pair per one (1) minute. The replication time should be prolonged gradually as replication is repeated, since the activity of Taq. Polymerase may be decreased, thus the replication time should be allowed sufficiently (10 minutes) in the final cycle to let the polymerase act thoroughly. The above processes are repeated (normally, 25 to 30 cycles) to amplify a desired part of DNA.
The Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), a similar reaction to the Polymerase Chain Reaction, is a process to amplify messenger ribonucleic acid (hereinafter, mRNA). This reaction is a process for replicating mRNA into complimentary DNA (cDNA) which then is amplified through the Polymerase Chain Reaction. This process is employed for searching a specific gene which cannot be detected from DNA, but can be detected only by RNA amplification.
Recently, the real-time Polymerase Chain Reaction has been known to the public. The real-time Polymerase Chain Reaction, is a technique which can monitor the process of the chain reaction in real-time, by means of measuring the intensity of the fluorescence emitted from reaction tube, which represents the progress of each cycle, without separation of the product of chain reaction on the gel. The real-time monitoring apparatus for this Polymerase Chain Reaction, is a machinery which is a combination of a thermal cycler for the Polymerase Chain Reaction and a fluorometer for detecting the reaction product. The conventional real-time monitoring apparatus for the Polymerase Chain Reaction, consists of a heat generator; thermal conduction block for heat transfer to a reaction tube containing reaction mixture; a light source to radiate light on reaction mixture in the tube; and a light receiving section for receiving fluorescence emitted from reaction mixture. The general scheme of real-time Polymerase Chain Reaction monitoring, comprises a step for repeating heating and cooling cycle by means of heat generator to proceed the reaction in a tube; a step for radiating light on the reaction mixture by using a light source at the end of the each cycle; and measuring the intensity of fluorescence emitted from the reaction mixture, to verify the progress of the Polymerase Chain Reaction.
However, the real-time monitoring apparatus for the Polymerase Chain Reaction which have been known to the public until now, have several problems that they cannot treat reaction mixture sequentially with a certain time interval whereas they can treat a large number of reaction mixture, and that other reaction mixture cannot be put into the apparatus until the reaction of the preceding mixture is completed. In addition, the conventional real-time monitoring apparatus should measure the intensity of fluorescence in each amplification cycle. Consequently, it provides a poor accuracy in measurement of the progress of the reaction, since measuring fluorescence of a number of reaction mixture takes a longer period of time.
Various real-time monitoring techniques of the Polymerase Chain Reaction have been studied and reported to overcome the problems of the conventional apparatus. The apparatus shown in FIG. 4 is similar to the apparatus of the present inventions amongst them.
FIG. 4 shows the Polymerase Chain Reaction machine based on the conventional technique (U.S. Pat. No. 6,033,880) which employs a capillary tube. The thermal conduction block (300) is composed of four (4) different types of constant temperature blocks (302, 303, 304, etc.). The reaction mixture and reagent are supplied into or removed from the capillary tube (351) by means of a reagent supplying apparatus (350). The thermal conduction rotates to change the temperature of the capillary tube and thus, the Polymerase Chain Reaction is performed. The common problems of this type of techniques are that it is required to rotate a thermal conduction block for the Polymerase Chain Reaction and that there may be a difference in the progress of the Polymerase Chain Reaction owing to the difference of contact between each capillary and thermal conduction block, which causes the decline of reproducibility.
In addition, it is unable to perform the Polymerase Chain Reaction continuously with a certain time interval in this type of apparatus. Moreover, there is a serious problem that it is unable to check the progress of reaction until the reaction is completed.
A novel apparatus for real-time monitoring of Polymerase Chain Reaction, which can overcome the problems of the conventional, has been introduced. J. H. Hahn et al. of Pohang University of Science and Technology proposed a temperature control and circulation apparatus for the continuous and stream-type Polymerase Chain Reaction which consists of a capillary and a round type thermal block (Korean Patent Application No. 10-2004-0006740, Feb. 4, 2004).
The above apparatus is composed of capillary tube in the length of 3.5 meters and coiled 33 rounds around the copper block of which a diameter is 30 millimeters, and which is made up of three temperature zones of melting, annealing and extension. Each cycle of PCR of the reaction mixture which flows within the capillary tube is proceeded upon each round around thermal block made from copper. (Nokyoung Park, Suhyeon Kim and Jong Hoon Hahn, Cylindrical Compact Thermal-Cycling Device for Continuous-Flow Polymerase Chain Reaction, Anal. Chem., 75, 6029-6033).
The process of Hahn et al. includes the step of coiling a heating block with capillary tube wherein the reaction mixture of Polymerase Chain Reaction flow; the step of radiating light on the capillary tube using a apparatus being located at a scanner; and the step of scanning the capillary tube using a scanning apparatus including a fluorometer to measure the intensity of the fluorescence generated from capillary tube.
In this process, a moving stage including a radiation part to radiate light on the capillary tube coiling around a heating block, and a sensor to measure the intensity of the fluorescence generated from the capillary tube moves the scanner linearly to radiate light on the capillary tube along with the path of the scanner, thus measuring the intensity of the fluorescence generated from the capillary tube.
The above patent of Hahn et al. describes a scanning apparatus which comprises a radiation part such as laser and lamp to generate light of a specific wavelength; and a fluorescence detector such as PMT and diode, wherein said radiation part and said fluorescence detector move as a constant speed on the heating block which is coiled with a capillary tube for scanning. A scanner having the radiation part and fluorescence detection sensors attached thereto moves along the axis which is parallel or perpendicular to the axis of the heating block coiled with a capillary tube, thus radiating light on the capillary tube or measuring the intensity of the fluorescence.
It is necessary to have a transporting apparatus such as a stepping motor and a linear transporting means, a guidance apparatus for transporting, and a driving means to provide power to a transporting apparatus, in order to have a portion for the fluorescence detection part and a fluorometer attached to a moving stage and move the moving stage at a constant speed, scanning and detecting the generated fluorescence continuously. The radiation and radiation part which uses one or more optical lenses should use not only the highly costed optical lenses such as substance lens, but also a delicate arrangement apparatus for precise adjustment and deviation of the light channel. These optical apparatuses which are used for radiation and to control and adjust the light channel are not only expensive but also take considerable space. Moreover, these driving means, power transmission means, transporting apparatus, and etc. also cause the problem of the space required for an apparatus for the Polymerase Chain Reaction, because these mechanical apparatuses and linear motor apparatuses make the Polymerase Chain Reaction machine large in size and may cause frequent breakdown and malfunction.
As considered above, one or more expensive lenses including a substance lens should be used to radiate light on the capillary tube that changes its location according to the movement of light source. Another cause of raising the production cost of the Polymerase Chain Reaction machine is the delicate mechanical processing on installation.
The apparatus of Hahn et al. monitors the Polymerase Chain Reaction whenever the scanner having a moving light source, a fluorescence detection part and one or more expensive optical lens apparatuses attached thereto scans one or more capillary tubes.
Such scanning method causes problems such as that light may be radiated during the scanning process, that it can only monitor the reaction within the capillary tube when the fluorometer passes and that it cannot monitor the reaction that occurs at the other part within the capillary tube where the scanner cannot cover.
Therefore, there is a need in the art to develop a new type of the real-time monitoring apparatus to overcome the chronic problems in Hahn et al. by providing a smaller in size, inexpensive, continuously performing, practical and effective real-time monitoring apparatus.